This invention concerns methods and apparatus for imaging, particularly the imaging of fluorescing samples of the type in which the sample is first illuminated with an excitation radiation such as ultra-violet light, and is subsequently interrogated for any resulting emission light due to fluorescence within the sample.
U.S. Pat. No. 4,922,092 describes a fibre optic device used to couple the light emitted from an array of well sites in a plate to an imaging device.
Our concurrent International Patent Application No. PCT/GB97/01825, filed Jul. 4, 1997, describes alternative arranges and systems incorporating fibre optic devices by which a large number of well sites can be inspected for fluorescence arising therefrom.
It is an object of the present invention to provide an improved optical fibre transfer device and an improved method of coupling the output of such a device to a camera input.
In FIGS. 10 and 11 of our concurrent Application, the output end of the fibre optic plate is shown coupled to a camera input window via a filter. The purpose of the filter is to restrict wavelengths entering the camera to those of expected, or wanted, emissions so as to ideally remove from the camera input any radiation at unwanted wavelengths such as stray excitation radiation transmitted via the fibre optic plate, or the like.
In an example involving 96 fibre optic bundles, the 96 outputs of the bundles may be arranged in any convenient configuration or aspect ratio depending on the output of the camera to which the image is to be applied. Where the latter is generally circular, the 96 rods may be arranged in a generally circular or hexagonal array so as to substantially fill the entrance window of the camera, and if the latter is 40 mm diameter and the rods fill an area of 32xc3x9742 mm21, there should be adequate spacing between rod centres (approximately 2.5 mm) to ensure minimal cross-talk between bundles. Where an emission filter is inserted between the plate and the camera input, this should be as thin as possible, and may need to be less than 0.5 mm to ensure acceptable levels of cross-talk.
Since the excitation wavelengths may be very close to the wavelengths of emitted radiation from the samples or where two or more emitted radiations can arise and selection as between one wavelength and another is desired, the difference between the wavelengths of the different emitted radiations may be very small. In these circumstances ordinary filters may not be sufficiently selective.
According to the present invention in an imaging system for fluorescence assays, a fibre optic coupling plate for transmitting radiation emitted by a sample towards a camera, is combined with an interference filter so as to enable highly selective transmission of radiation to the camera, according to wavelength.
The present invention also envisages the combination of an interference filter with a modified fibre optic coupling plate such as described herein, in which the sample sites or wells of an array thereof are viewed by separate fibre optic bundles, and each bundle transmits emitted light from a one sample or well to a discrete region of the field of view of the camera.
Typically the interference filter is located between the output end of the fibre optic coupling plate and the camera input window.
Where a system is intended to operate at a single wavelength, the interference filter may form part or comprise the input window of the camera.
Where, as is more likely, the system is to be capable of handling different assay chemistries and selection of different wavelengths, provision may be made for interchanging the filter so as to render the camera more or less sensitive to different wavelengths.
In one arrangement, two or more interference filters may be located in apertures in a slidable or rotatable support plate located between the output side of the fibre optic coupling plate and the input window of the camera, and the filter support plate filters is movable so as present one or another of the filters to the camera as appropriate.
The movement may be effected manually, or drive means may be provided, to effect the movement to position different filters in place.
Where the inspection system is computer controlled, the control system may be programmed to move the filter support plate according to the assay chemistry and/or wavelengths involved, or may be programmed so as to present a sequence of different filters during the course of inspection of each set of well sites, and the camera output is switched or flagged accordingly, so as to allow the different filters to be linked to the different camera output signals.
Use of an interference filter instead of an absorption filter allows improved discrimination of emission wavelengths and the improved blocking of unwanted wavelengths, whether residual excitation reduction or other emissions arising from the excitation.
The use of an interference filter may however introduce two problems.
Such a filter is quite thick (eg 5-10 mm), which can lead to cross talk between well emissions due to loss of spatial resolution. Furthermore, the central wavelength of the bandpass range shifts with angle of incidence eg at 15xc2x0 from the normal to the filter, the shift is typically 4 nm. This may be acceptable in many instances but at 30xc2x0, the shift is typically 16 nm, which is almost certainly unacceptable.
One solution is to compromise light gather efficiency by using fibre optic bundles having smaller numerical aperture of say 0.6 instead of 1. However this will not limit the angle of extreme rays (sinxe2x88x921 0.6=37).
Since it is a primary requirement of systems such as described herein, (and. in our concurrent Application) that fibre optic bundles are used having a high numerical input aperture (ie angular acceptance), the numerical aperture of the output end of the fibre optic bundle will also be high. This results in a large cone of rays emanating from the end of each fibre towards the filter. This is generally incompatible with the input requirements of an interference filter which operate best when input rays have only a small angular spread.
According therefore to a further feature of the present invention, optical means is incorporated which effectively reduces the numerical aperture of the output of the coupling plate as seen by the interference filter.
In a preferred arrangement, optical means is provided which cause the light emitted from the coupling plate to be converted into a parallel beam.
In one arrangement magnifying optical means is provided between the output face of the coupling plate and the interference filter so as to present an enlarged image of the coupling plate output face, to the interference filter.
Typically a separate lens is required for each fibre optic bundle. In such an arrangement a mini-lens or a gradient index (GRIN) lens may be placed at the end of each bundle of optical fibres.
Disadvantages of a lens approach include wavelength dependence of the optics, vignetting, limited acceptance numerical aperture, difficulty of restricting angular range of skew rays or rays coming from the edges of the optical fibres and the need for careful alignment of lenses with the fibres.
Alternatively and more preferably, the fibres making up the bundles may increase in cross-section as between the input and output ends of the plate, so as to present an enlarged image of the well emissions to the interference filter.
According to a preferred feature of the invention, therefore a preferred solution involves the use of a fibre optic coupling plate in which the fibres taper in cross-section from the output end to the input end so that the area of each fibre and therefore each fibre optic bundle in the output face of the plate is greater than that of the particular fibre or fibre optic bundle in the input face of the plate.
Typically the size ratio between the input and output ends of the plate is 15.45.
Typically the diameter of the camera input is 45 mm and a well plate has 96 wells, which therefore require there to be 96 fibre optic bundles. Conveniently the 96 fibre optic bundles are arranged in a hexagonal array in the output face of the coupling plate. The input diameter end of the bundles is typically 0.6 mm, and these are typically arranged with a 1 mm spacing. The angular spread of rays leaving a bundle and entering an interference filter thus will be reduced to one third of that of rays entering the end of the bundle, and will typically be of the order of 120, so reducing the wavelength shift to only 3 nanometres. With a 7 mm thick interference filter there should be negligible cross-over of rays between adjacent fibre optic bundles, since the output ends of the bundles will have a diameter of 1.8 mm.
In either event (using lenses or tapering fibres), the larger image presented to the interference filter results in a smaller angular spread of light rays so that the interference filter is able more readily to function and discriminate between one wavelength and another.
As mentioned previously, preferably a small air gap exists on opposite sides of the interference filter to enable it to be removed and replaced with a different interference filter as required, for example by means of a filter wheel. By keeping the air gaps small typically in the range 0.1 to 0.2 mm, negligible spreading of light from the ends of the fibre optic bundles will occur.
The preferred arrangement avoids most of the problems associated with mini lenses or GRIN lenses. There is negligible wavelength dependence, little vignetting, high input numerical apertures are readily obtained, and such problems as remain (in general relatively small by comparison) extreme stray and skew rays and geometrical alignment.
According to a further preferred feature of the invention, in addition to the use of a magnifying fibre optic plate as aforesaid, a further optical device may be used to advantage between the output end of the magnifying coupling plate and the camera input so as to substantially exclude extreme stray and skew rays from the input to the camera.
In a preferred arrangement the image formed on the output of the magnifying coupling plate may be focused onto the camera input by means of an optical lens. Since the angular spread of non-extreme rays of light from any point on the coupling plate output face will be 12xc2x0 or less, (measured in air), most of the light leaving the output face of the plate can be collected by a lens having an acceptance aperture of f1.2. Such a lens is readily constructed typically as a multi-element lens and the advantage is that rays greater than 12xc2x0 such as extreme, stray and skew rays will not be collected by the lens. The presence of the lens therefore will tend to eliminate cross-talk between light emanating from the output of adjacent fibre optic bundles and light with large wavelength shifts. Furthermore it will also facilitate the manipulation of interference filters since a larger air gap between coupling plate and filter can be permitted.
Where the diameter of the faceplate and camera inputs have been matched, the lens preferably has unity magnification.
Where any mismatch exists between the output face of the coupling plate and the camera input, this can be accommodated by using a lens of appropriate magnification.
Alternatively the optical lens may be replaced by a second fibre optic coupling plate in which separate fibre optic bundles are held in a spaced array to define an input face in which the size, spacing and arrangement of the fibre optic bundle ends corresponds to those forming the output face of the coupling plate between the interference filter and the reaction sites, and wherein the opposite ends of the fibre optic bundles are arranged so as better to conform to the camera input size and/or aspect ratio. Where the latter has a rectangular field of view, the output ends of the fibre optic bundles may be arranged in a rectangular matrix of appropriate dimensions so as to be accommodated within the input window of the camera.
The optical fibres making up the bundles in the second coupling plate may vary in cross-section from input to output so as to present either an enlarged or a reduced size image or may be of constant cross-section so that there is no magnification or demagnification between input and output of the second coupling plate.
Where any lens or other optical element is employed, preferably this is corrected chromatically for the wavelengths expected to be handled by it.
Where two or more wavelengths are to be investigated requiring two or more filters it may be simpler and cheaper to construct matching pairs of interference filter and lens, (or interference filter and second coupling plate), each element of each pair being chromatically corrected for the wavelength(s) it is intended to be used with and each filter and lens (or coupling plate), is mounted in a cylindrical passage extending between two faces of a support member adapted to be moved, (such as rotated) so as to present first one and then another of the pairs of elements in the path between the first coupling plate output and the camera input.
Where air gaps are required between fibre optic bundle ends and interference filters and the like, it has been found that air gaps of up to 0.5 mm can be accommodated, although for many reasons smaller air gaps are preferred.
According to a further aspect of the invention, the primary coupling plate may be constructed quite differently so as to avoid the need for the sequential presentation of different interference filters between the output and the camera. In this arrangement non-tapering optical fibres (or fibres in which the taper is reversed so that each fibre output end is smaller than its input end) may be used. The output end of each bundle of fibres is well spaced from adjoining fibre optic bundle ends and an interference filter having an appropriate central (or peak) wavelength is positioned between the output face of the coupling plate and the camera so that emissions of different wavelength from a fibre optic bundle appear at different diameters on the output face of the filter relative to the position of the centre or peak wavelength emissions from the relevant optical fibre bundle.
Provided the pixel resolution of the camera is high enough, the different diameter rings of light corresponding to the different wavelengths emanating from each fibre optic end can be identified and measured using standard image processing techniques.
It is an advantage of such a method that where different wavelengths are expected from an assay, and both or all need to be checked before the analysis can be completed, all of the wavelengths emissions can be checked during a single inspection period, instead of during a succession of such periods.
Where the assay period is for example 10 minutes or longer, the saving in time if (say) three different wavelengths are to be investigated, is considerable.
According to a further aspect of the invention the transmission of skew rays arising in the apparatus is reduced by substituting an angle collimating plate for the lens or second coupling plate.
The collimating plate may be constructed from a plurality of optical fibres arranged in a bundle, each of which has a low numerical aperture (NA).
A typical NA for each optical fibre would be 0.2.
The optical fibres making up the bundle are preferably fused into a unitary plate.
The plate is preferably located just in advance of the fibre optic input plate of the camera.
Alternatively the plate may be integrated into or comprise the fibre optic camera input plate.
According to still another aspect of the invention, multiple wavelength emission analysis of fluorescent arrays producing up to N different wavelength emissions is achieved by providing a fibre optic device having input and output faces between the fluorescing samples and the interference filter which divides the emitted light into N different paths, each of which presents its fraction of the emitted light over one of N different discrete regions in the output face of the device, and N different interference filters are located in the light path between the output face and the camera input faceplate, each registering with one of the said N discrete regions in the output face of the device, and each of the N filters being selective of a unique one of the N different wavelengths which can arise in the emitted light.
Each of the N different paths in the device nay be made up of one or a plurality of optical fibres.
The cross-section of each fibre path or number of fibres in each path, may be the same for each of the said N paths, or may be different. Where light of one wavelength is emitted at a low level, larger areas of fibres (or number of fibres) may be provided for supplying light to the relevant discrete regions and associated filter, for that wavelength, to ensure there is a sufficient quantity of light of that wavelength at the camera input, to register.
Where the quantity of light of different wavelengths is to be compared one with another, so as for example to allow a ratio (or series of ratios) to be obtained, each of the N light paths should have substantially the same light transmission characteristics (disregarding the filter), but where this is not the case, or where the light capturing and transmitting capabilities of one or more paths is enhanced relative to the others, a scaling factor may be determined and stored for adjusting the camera output signals relating to the received light from each such enhanced path.
The different paths and filters may have differing light transmitting characteristics which may be inherent or due to manufacturing tolerances, or both. To this end the camera is preferably calibrated using standard light emitting devices producing known levels of light emission at selected wavelengths, and a look up table of scaling factors is created for adjusting the camera output signals during subsequent analysis scans, so as to normalise the output signals corresponding to the different paths and thereby allow a more accurate assessment of the relative values of the different wavelengths to be obtained.
N will usually only be 2 or 3, although the invention is not limited to such small numbers of different paths.